1. Field of the Invention
The present invention deals with a beam splitting device in particular for a laser scanning microscope, comprising a laser producing a laser beam and a system of intentionally arranged partially and totally reflecting mirrors splitting the laser beam into multiple partial beams, whereby the partial beams leave the beam splitter device in a plane and are fed to the sample under investigation.
2. Description of the Prior Art
From the previous art (DE 196 53 413 A1) it is known that a multitude of partial beams can be produced by allowing an expanded laser beam to be incident on a micro-lens array.
This means in particular, that each partial beam is generated from a different spatial part of the beam profile of the incident beam. The partial beams are focused by the micro lenses according to their focal length in one or more closely spaced planes behind the micro lens array and propagate towards the microscope objective lens which focuses the partial beams onto the sample. Hereby the beams over-illuminate the aperture of the microscope objective lens. The number and distance between the partial beams is dictated by the design of the micro-lens array.
Typically in these optical devices there is a bifurcation of the optical path between the light source for the laser beam and the sample in order to separate and direct the fluorescence originating from the sample towards a photodetector. A dichroic mirror is used to separate and direct the fluorescence light towards the photodetector.
A problem of the known beam splitter is that the individual partial beams have substantially different intensities. This is due to the fact that laser beams have a distribution of intensity across the beam cross-section. Lasers used in laser scanning microscopy typically have a gaussian beam profile. This means, the partial beams that are generated by a micro-lens array from the centre of the laser beam are more intense than those beams generated from the edge of the expanded beam. The detected signal is the fluorescence excited by the partial beams.
In the case of one photon excitation this signal is proportional to the intensity of the exciting partial beams. In non-linear microscopy (2, 3 or more photon excitation) this effect is squared or cubed respectively. For example for two beams differing a factor of 4 in intensity the signals generated by these beams differ by a factor of 4 (1 photon excitation), 16 (2 photon exc.) or 64 (3 photon exc.) . This example illustrates the problems when one uses micro-lens arrays for the generation of multiple partial beams especially in conjunction with non-linear laser scanning microscopy.
Another problem of the known microscope is that the resolution so obtained is not as good as allowed by the diffraction limit of the employed microscope objective lens.
Lateral resolution in laser scanning microscopy is given by the full width at half maximum of the focus generated in the sample. In the case of the known microscope the size of the focus is not only determined by the aperture of the employed microscope objective lens but also by shape and size of the micro lenses because the micro lenses act as the limiting diffracting aperture for every partial beam. This means in particular, that the resolution decreases with increasing number of beams because the size of the micro lenses is reduced and the diameter of the foci rises.
Furthermore, the micro lenses are not illuminated symmetrically and more so with outer part of the beam profile of an expanded laser beam generally decreasing towards the edge. As a consequence the foci have an asymmetric intensity distribution in the sample.
As resolution is one of the most important parameters in microscopy this is not an irrelevant problem.
Yet another problem of the beam splitter is that the energy of the incident laser beam is not used completely.
Part of the expanded laser beam is cut off by the edge of the micro lens array and by the edges of the individual micro lenses. These losses add up so that e.g. starting from 1.4 W output power of the laser only about 125 mW (9%) reach the sample (Jorg Bewersdorf, Rainer Pick, Stefan W. Hell, "Multifocal multiphoton microscopy", Optics Letters, Vol. 23, No. 9 (1998).)
The loss of laser power substantially reduces optical efficiency of the microscope because the required time for a measurement depends on the amount of laser power at the sample. This is especially of importance in non-linear microscope systems where the excitation efficiency depends non-linear on the excitation intensity.
For biological applications of laser scanning microscopy where processes in living cells are studied the required time for a measurement is an important aspect because it determines the maximum speed with which the processes can be investigated. The study of the propagation of impulses in nerve cells is an example (R. Yuste, W. Denk, "Dendritic spines as basic functional units of neuronal integration", Nature Vol. 375, pp. 682-684 (1995).) Here laser scanning microscopy was required to obtain high-resolution images from deep layers of intact nervous tissue The slow imaging speed prevented the study of the propagation of impulses with two-dimensional spatial resolution. An enhancement in the imaging speed will lead to the further extension of laser scanning microscopy to faster processes.
Crucial for imaging speed is the laser power inside the sample. As the power of a single beam can not be increased arbitrarily without damaging the sample, the power inside the sample can only be raised substantially by illuminating several points using several beams simultaneously. Here the efficacy of the beam splitting method determines (for a fixed output power of the laser) the maximum number of partial beams.
Another problem of the known microscope is that distance and number of partial beams are fixed by the nature of the micro-lens array. Depending on the application, a smaller or larger distance between the beams is desirable.
Single cells can be studied with maximum speed if all beams are directed at the one cell under investigation. In this case a distance of approx. 2 .mu.m between the beams is reasonable.
If microstructured substrates for the analysis of bio-chemical reactions ("biochips") are to be analysed, a distance on the order of the microstructure is desired(approx. 20 .mu.m). The beam splitter of the known art does not offer the possibility of changing number and distance between beams.
In DE 39 18 412 A1, another beam splitting device for a laser scanning microscope is described comprising a laser producing a laser beam and a semi-transparent mirror dividing the said laser beam into partial beams, said partial beams leaving the beam splitting device in a plane and being guided to the sample under investigation.
In DE 195 35 525 A1 a beam splitter is described comprising a semi-transparent and a highly reflective layer, said semi-transparent layer being located in the optical path in front of said highly reflective layer and reflecting back to said highly reflective layer and having a transmission increasing with the number of reflections in order to get partial beams with equal intensity.
In EP 0 386 643 A2 a beam splitter is described comprising a semi-transparent and a highly reflective layer, said semi-transparent layer being in front of said highly reflective layer and in different embodiments being tilted or stepped and where in another embodiment two beam splitters tilted by 90 degrees with respect to each other are provided in order to generate a two-dimensional illumination array.
In DE 38 76 344 T2 an optical beam divider is described comprising a semi-transparent layer having a constant transmission and being located between to highly reflective layers, said layers being parallel to each other and said semi-transparent layer transmitting the incident light to one of said highly reflective layers and reflecting the light to the other one of said highly reflective layers with equal attenuation and again and repeatedly transmitting the light to one of said highly reflective mirrors and reflecting it to the other one of said highly reflective mirrors with equal attenuation.